The instant invention is in the field of methods for chemical analysis of biological material and more specifically the instant invention is in the field of methods for determining chemically related differences between subject biological material and control biological material by way of separation techniques such as chromatography.
It is common practice to extract various chemical compounds from biological materials (such as leaves and/or stems and/or seeds from a plant) by contacting the biological material with a fluid extractant such as hexane or water. The extract may then be chromatographed, for example, by gas chromatography or liquid chromatography, to produce a chromatogram. The chromatogram indicates the separation and detection of the extracted chemical compounds.
Existing chemical analysis methods for analyzing subject biological material often are directed towards the determination of specific target chemical compound(s). For example, a plant gene may be modified so that the seeds of the plant yield an elevated level of a specific amino acid such as lysine. In this case the chemical analysis method used would target lysine.
However, when an organism is, for example, genetically modified without knowing what changes may result in the chemistry of the organism, then a chemical analysis method is needed which is capable of determining a broad range of chemical compounds. This kind of genetic modification is used, e.g., to determine the activity encoded by a new gene such as may be obtained by techniques well known in the molecular biology art such as gene discovery, gene recombination, gene mutagenesis, and stochastic gene synthesis (i.e. gene formation by random linkage of, e.g., nucleotides, trinucleotides, or secondary-structure-encoding oligonucleotides).
For example, in regard to gene discovery, about 46% of naturally occurring genes sequenced to date by The Institute for Genomic Research TIGR, Rockville, Md.) are of unknown functionxe2x80x94being totally new to biologyxe2x80x94and about 50% of these are thought to be of broad biological Importance as they have been found conserved among diverse species (according to J. Craig Venter, TIGR Chairman, xe2x80x9cDecoding the Human Genome,xe2x80x9d a public lecture given May 24, 1999 in Midland, Mich.). Defining the nature of such genes, as they function in vivo, would require an efficient method for identifying the in vivo effectsxe2x80x94and thus the, e.g., regulatory, biocatalytic, or transductional activitiesxe2x80x94of the expression products of such genes. Such a method would need to be capable of quickly comparing the concentrations of multiple metabolite species present in cells or organisms modified to contain such genes against the concentrations of multiple metabolite species present in unmodified cells or organisms.
It would be an advance in the art of chemical analysis if a method were developed for determining chemically related differences between subject biological material and control biological material, which method would be capable of determining a broad range of chemical compounds and which method preferably would be capable of rapid and automated use.
The instant invention provides a method for determining chemically related differences between subject biological material and control biological material which method is capable of determining a broad range of chemical compounds and capable of rapid, automated use. More specifically, the instant invention is a chemical analysis method for determining chemically related differences between subject biological material and control biological material, which method comprises at least the following six steps. The first stop is to contact the subject biological material with a fluid extractant to produce an original fluid extract of the subject biological material. The second step is to contact the control biological material with the fluid extractant to produce an original fluid extract of the control biological material. The third step is to chromatograph the fluid extract of the subject biological material, to produce a chromatogram of the fluid extract of the subject biological material. The fourth step is to chromatograph the fluid extract of the control biological material to produce a chromatogram of the fluid extract of the control biological material. The fifth step is to determine the differences between the chromatograms of the third and fourth steps to identify at least one outlier peak. The sixth step is to determine the chemical identity of the outlier peak, for example, using gas chromatography/mass spectroscopy antis of the outlier peak.